The goal of this project is to understand the pathway by which intracellular proteins of Salmonella tyohimurium are degraded. New knowledge concerning this pathway should contribute to an increased understanding of the physiological significance of intracellular proteolysis and should be useful in elucidating similar proteolytic processes that exist in all types of cells. Three lines of experimentation will be pursued: 1. A system will be developed in which the degradation products of a single protein can be observed. The gene for the protein to be studied will be cloned behind a phage T7 promoter so that the protein can be expressed and radiolabeled in the absence of other protein synthesis. The pattern of intermediates in the degradation of this protein and the effect of mutations in protease genes on this pattern will be studied. The specificities of the exopeptidases already shown to function on the pathway of protein degradation will be determined using purified enzymes and appropriate peptide substrates including the intermediates identified in in vivo experiments. The results of these studies should define where in the degradation pathway these exopeptidases function. 2. Genes coding for endoproteases that may act in the early steps of the degradation pathway will be identified and inactivated or overexpressed by mutation. The effects of these mutations on the pattern of intermediates observed in 1. will be determined to define the role of each enzyme in the pathway. 3. The regulation of the genes coding for important components of the proteolytic apparatus will be studied. The nature of the signals that determine the levels of expression of these genes may provide important clues about the physiological significance of intracellular proteolysis.